The basic objectives of this research proposal are to isolate and characterize the gene for a lysosomal enzyme, to determine the structure of the enzyme, and to study factors governing gene expression in order to elucidate the basis of inherited lysosomal storage disorders. The lysosomal enzymes share a common location and type of function within the cell. They also exhibit some similar structural features. A deficiency of one or more of these enzymes is responsible for the group of inherited human diseases known as the lysosomal storage disorders. Little is currently known about the structures of genes coding for these enzymes and genomic elements which may regulate their expression. Such information is important for designing a successful therapy for the lysosomal storage disorders. A complete amino acid sequence has yet to be determined for any enzyme of the group, and many question about the synthesis and post-translational modifications of these enzymes remain to be answered. Beta-glucuronidase is the most thoroughly studied of the acid hydrolases. In addition to its lysosomal location in most tissues, it has been found in association with the endoplasmic reticulum of liver and kidney. Evidence suggests that the enzyme found at both sites is specified by the same structural gene. The long range goals of this project include analysis of portions of genomic DNA which code for beta-glucuronidase. Initially, this will involve preparation of cDNA cones from rat preputial gland Beta-glucuronidase mRNA. These will be used to select similar sequences from rat and human genomic DNA. The rat and human DNAs will be sequenced and compared to define structural gene and possible transcriptional control regions in the DNA. An amino acid sequence will be proposed for the rat and human enzymes, and structural features related to post-translational modifications of the enzymes sought. Gene expression will be studied in fibroblasts from normal individuals and from those with an inherited deficiency of Beta-glucuronidase. The project will be designed to define the nature of inherited lysosomal storage disorders and to provide basic information needed to develop a rational therapy for these disorders.